Metabolic Engineering Tools Research Articles

Paired-termini antisense RNA mediated inhibition of DoxR in Streptomyces peucetius ATCC 27952

Our previous study provided an insight into DoxR as a negative regulator of doxorubicin production in Streptomyces peucetius ATCC 27952. Streptomyces hosts are advantageous in terms of producing a number of pharmaceuticals in low titer. Antisense RNAs (asRNAs) silencing strategy acts as an alternative tool for metabolic engineering of microorganisms for construction of an efficient cell factory. In this study, a paired-termini antisense RNAs (PTasRNAs) silencing strategy was employed for inhibition of DoxR to enhance doxorubicin production. To continue this endeavor, we designed and constructed the piBR702 vector for the expression of PTasRNAs in monocistronic mode. Further, two variants of asRNA, adoxR and bdoxR were designed and cloned into piBR702. All the rDNAs were transformed into S. peucetius to generate engineered strains. The engineered strains, S. peucetius A and S. peucetius B produced enhanced titers of doxorubicin, daunorubicin, and e-rhodomycinone; however, no such change was seen in S. peucetius AB. Moreover, RT-PCR analysis of doxR from S. peucetius A and S. peucetius B, together with the higher production from S. peucetius A, confirmed adoxR as a better asRNA than bdoxR. The reason behind this could be due to the simple secondary structure and low binding free energy of adoxR (−419 kcal/mol) than bdoxR (−358.8 kcal/mol). Our study demonstrated that antibiotic production was enhanced significantly by inhibiting DoxR, a negative regulator in S. peucetius using PTasRNAs. In addition, this study further provides an insight into PTasRNAs as an effective tool for gene silencing in Streptomyces and its use as an effective tool for metabolic engineering.

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

AbstractAn immense number of chemical reactions are carried out simultaneously in living cells. Nature’s optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man‐made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor‐made microbial cell factories.magnified image

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Genome-scale strain designs based on regulatory minimal cut sets.

Stoichiometric and constraint-based methods of computational strain design have become an important tool for rational metabolic engineering. One of those relies on the concept of constrained minimal cut sets (cMCSs). However, as most other techniques, cMCSs may consider only reaction (or gene) knockouts to achieve a desired phenotype. We generalize the cMCSs approach to constrained regulatory MCSs (cRegMCSs), where up/downregulation of reaction rates can be combined along with reaction deletions. We show that flux up/downregulations can virtually be treated as cuts allowing their direct integration into the algorithmic framework of cMCSs. Because of vastly enlarged search spaces in genome-scale networks, we developed strategies to (optionally) preselect suitable candidates for flux regulation and novel algorithmic techniques to further enhance efficiency and speed of cMCSs calculation. We illustrate the cRegMCSs approach by a simple example network and apply it then by identifying strain designs for ethanol production in a genome-scale metabolic model of Escherichia coli. The results clearly show that cRegMCSs combining reaction deletions and flux regulations provide a much larger number of suitable strain designs, many of which are significantly smaller relative to cMCSs involving only knockouts. Furthermore, with cRegMCSs, one may also enable the fine tuning of desired behaviours in a narrower range. The new cRegMCSs approach may thus accelerate the implementation of model-based strain designs for the bio-based production of fuels and chemicals. MATLAB code and the examples can be downloaded at http://www.mpi-magdeburg.mpg.de/projects/cna/etcdownloads.html. krishna.mahadevan@utoronto.ca or klamt@mpi-magdeburg.mpg.de Supplementary data are available at Bioinformatics online.

Identifying promoters for gene expression in Clostridium thermocellum

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase (lacZ) and NADPH-alcohol dehydrogenase (adhB) in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB).

Combinatorial and high-throughput screening approaches for strain engineering.

Microbes have long been used in the industry to produce valuable biochemicals. Combinatorial engineering approaches, new strain engineering tools derived from inverse metabolic engineering, have started to attract attention in recent years, including genome shuffling, error-prone DNA polymerase, global transcription machinery engineering (gTME), random knockout/overexpression libraries, ribosome engineering, multiplex automated genome engineering (MAGE), customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER), and library construction of "tunable intergenic regions" (TIGR). Since combinatorial approaches and high-throughput screening methods are fundamentally interconnected, color/fluorescence-based, growth-based, and biosensor-based high-throughput screening methods have been reviewed. We believe that with the help of metabolic engineering tools and new combinatorial approaches, plus effective high-throughput screening methods, researchers will be able to achieve better results on improving microorganism performance under stress or enhancing biochemical yield.

Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.

A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.

Altered Levels of Aroma and Volatiles by Metabolic Engineering of Shikimate Pathway Genes in Tomato Fruits

The tomato (Solanum lycopersicum) fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9) and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12) were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of fruits aroma and the generation of new combinations of tomato flavors.

A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

Heterotrophic growth of microalgae: metabolic aspects.

Microalgae are considered photoautotrophic organisms, however several species have been found living in environments where autotrophic metabolism is not viable. Heterotrophic cultivation, i.e. cell growth and propagation with the use of an external carbon source under dark conditions, can be used to study the metabolic aspects of microalgae that are not strictly related to photoautotrophic growth and to obtain high value products. This manuscript reviews studies related to the metabolic aspects of heterotrophic grow of microalga. From the physiological and metabolic perspective, the screening of microalgal strains in different environments and the development of molecular and metabolic engineering tools, will lead to an increase in the number of known microalgae species that growth under strict heterotrophic conditions and the variety of carbon sources used by these microorganisms.

Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering.

Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.

From random mutagenesis to systems biology in metabolic engineering of mammalian cells

Metabolic engineering is rapidly developing, with a continuous stream of technological developments being employed to expand the portfolio of molecules produced in cell factories. For chemical production (e.g., amino acids, biofuels, among others), metabolic engineering has progressed through three phases [1]. Initially, biological products were obtained through random mutagenesis of production strains and large screening efforts. Improved microbial strains could be isolated, but mechanisms underlying the desired phenotype were often poorly understood [2]. Diverse molecular biology techniques facilitated the second phase, in which simple, intuitive modifications were made. The third phase now employs systems biology techniques to understand the effect of modifications on all other metabolic pathways and on cell physiology. Thus, we have entered an era in which metabolic engineering aims to improve microbial strains in a reproducible fashion, using complex designs based on detailed biochemical knowledge and computational model simulations. Here, we highlight the historical progression toward using systems biology in microbial metabolic engineering and compare this to the current status of mammalian production cell line development. Finally, we discuss the unique challenges in engineering mammalian cell lines for biotherapeutic production and outline how systems biology can facilitate metabolic engineering efforts for these platforms. The systems biology approach to metabolic engineering has been enabled by three primary advancements: whole-genome sequencing, gene editing tools and genome-scale models of cellular metabolism. The completion of the Escherichia coli K-12 genome sequencing effort in 1997 [3] provided a comprehensive parts list for targeted metabolic engineering and expanded the scope of our understanding of the machinery within this microbe. The further development of efficient genetic modification systems, such as the lambda Red recombination system [4], enabled the deployment of targeted metabolic engineering designs, such as the removal of competing pathways that divert flux away from the formation of a desired product. Predictions of the systemic effects of genetic modifications were enabled when the information in the sequenced genome was harnessed for the development of genome-scale models of metabolism [5]. These models contain all known biochemical reactions in a cell, thus allowing one to predict the overall impact of modifications on phenotypic traits such as growth rate and small molecule secretion. Systems biology approaches are now important tools in microbial metabolic engineering. Yim et al. genetically modified E. coli to produce 1,4-butanediol (BDO) by introducing heterologous genes to allow Hooman Hefzi Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA

New trends in biotechnological production of rosmarinic acid.

Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyl lactic acid, is widely distributed in the plant kingdom. Interest in it is growing due to its promising biological activities, including cognitive-enhancing effects and slowing the development of Alzheimer's disease, cancer chemoprotection or anti-inflammatory activity, among others. In order to meet the increasing demand for this compound, several biotechnological approaches to its production based on plant cell and hairy root cultures have been developed. Empirical strategies are currently being combined with metabolic engineering tools to increase RA production in plant cell platforms in a more rational way. Discussed here are the latest advances in the field, together with recent trends in plant biotechnology, such as the application of single use technology and the use of biosensors in downstream processes.

Reduction in carotenoid levels in the marine diatom Phaeodactylum tricornutum by artificial microRNAs targeted against the endogenous phytoene synthase gene.

MicroRNAs (miRNAs) are key regulators of gene expression in eukaryotes where they can function to downregulate expression levels or functioning of messenger RNAs (mRNAs) that are targeted by mature miRNAs displaying sequence homology. The 'active' mature miRNA forms are short RNAs which are processed from longer precursor miRNA molecules that have a stem-loop structure. While artificial miRNAs have been developed for gene knockdown experiments in a range of eukaryotes, it is not known whether artificial or endogenous miRNAs can functionally knockdown mRNA levels in the model marine diatom Phaeodactylum tricornutum. Here, we investigate the potential use of artificial microRNAs (amiRNAs) for targeted gene knockdowns in P. tricornutum, by generation of transformants harbouring a transgene cassette for the generation of amiRNAs designed to target the endogenous phytoene synthase (PSY) gene. In P. tricornutum, the amiRNA stem-loop precursor was processed to produce a mature amiRNA that successfully targeted the PSY mRNA and reduced PSY mRNA levels. As the PSY gene is a key component of the carotenoid biosynthetic pathway, the levels of carotenoids in the P. tricornutum amiRNA knockdown lines were reduced relative to untransformed control lines. This study demonstrates that artificial miRNAs can be successfully deployed for gene knockdown experiments in the model diatom P. tricornutum, providing a powerful tool for future metabolic engineering and synthetic biology experimentation in this model marine diatom.

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops.

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.

Recent applications of synthetic biology tools for yeast metabolic engineering.

The last 20 years of metabolic engineering has enabled bio-based production of fuels and chemicals from renewable carbon sources using cost-effective bioprocesses. Much of this work has been accomplished using engineered microorganisms that act as chemical factories. Although the time required to engineer microbial chemical factories has steadily decreased, improvement is still needed. Through the development of synthetic biology tools for key microbial hosts, it should be possible to further decrease the development times and improve the reliability of the resulting microorganism. Together with continuous decreases in price and improvements in DNA synthesis, assembly and sequencing, synthetic biology tools will rationalize time-consuming strain engineering, improve control of metabolic fluxes, and diversify screening assays for cellular metabolism. This review outlines some recently developed synthetic biology tools and their application to improve production of chemicals and fuels in yeast. Finally, we provide a perspective for the challenges that lie ahead.

Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content.

Flavonoids, due to their pharmacological attributes, have recently become target molecules for metabolic engineering in commonly consumed food crops. Strategies including expression of single genes and gene pyramiding have provided only limited success, due principally to the highly branched and complex biosynthetic pathway of the flavonoids. Transcription factors have been demonstrated as an efficient tool for metabolic engineering of this pathway, but often exhibit variation in heterologous systems relative to that in the homologous system. In the present work, Arabidopsis MYB transcription factor, AtMYB111, has been expressed in tobacco to study whether this can enhance flavonoid biosynthesis in heterologous system. The results suggest that AtMYB111 expression in transgenic tobacco enhances expression of genes of the phenylpropanoid pathway leading to an elevated content of flavonols. However, dark incubation of transgenic and wild type (WT) plants down-regulated both the expression of genes as well as flavonoid content as compared to light grown plants. The study concludes that AtMYB111 can be effectively used in heterologous systems, however, light is required for its action in modulating biosynthetic pathway.

Application of new metabolic engineering tools for Clostridium acetobutylicum.

The renewed interests in clostridial acetone-butanol-ethanol (ABE) fermentation as a next-generation biofuel source led to significantly intensified research in the past few years. This mini-review focuses on the current status of metabolic engineering techniques available for the model organism of ABE fermentation, Clostridium acetobutylicum. A comprehensive survey of various application examples covers two general issues related to both basic and applied research questions: (i) how to improve biofuel production and (ii) what information can be deduced from respective genotype/phenotype manipulations. Recently developed strategies to engineer C. acetobutylicum are summarized including the current portfolio of altered gene expression methodologies, as well as systematic (rational) and explorative (combinatorial) metabolic engineering approaches.

Trade-offs in engineering sugar utilization pathways for titratable control.

Titratable systems are common tools in metabolic engineering to tune the levels of enzymes and cellular components as part of pathway optimization. For nonmodel microorganisms with limited genetic tools, inducible sugar utilization pathways offer built-in titratable systems. However, these pathways can exhibit undesirable single-cell behaviors that hamper the uniform and tunable control of gene expression. Here, we applied mathematical modeling and single-cell measurements of l-arabinose utilization in Escherichia coli to systematically explore how sugar utilization pathways can be altered to achieve desirable inducible properties. We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration. For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway. Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter. Equivalent modifications to the d-xylose utilization pathway yielded similar responses, demonstrating the applicability of our observations. Overall, our findings indicate that there is no ideal set of typical alterations when co-opting natural utilization pathways for titratable control and suggest design rules for manipulating these pathways to advance basic genetic studies and the metabolic engineering of microorganisms for optimized chemical production.

Biohydrogen production from engineered microalgae Chlamydomonas reinhardtii

The green microalgae Chlamydomonas reinhardtti is well-known specie in the terms of <TEX>$H_2$</TEX> production by photo fermentation and has been studying for a long time. Although the <TEX>$H_2$</TEX> production yield is promising; there are some bottlenecks to enhance the yield and efficiency to focus on a well-designed, sustainable production and also scaling up for further studies. D1 protein of photosystem II (PSII) plays an important role in photosystem damage repair and related to <TEX>$H_2$</TEX> production. Because Chlamydomonas is the model algae and the genetic basis is well-studied; metabolic engineering tools are intended to use for enhanced production. Mutations are focused on D1 protein which aims long-lasting hydrogen production by blocking the PSII repair system thus <TEX>$O_2$</TEX> sensitive hydrogenases catalysis hydrogen production for a longer period of time under anaerobic and sulfur deprived conditions. Chlamydomonas CC124 as control strain and D1 mutant strains(D240, D239-40 and D240-41)are cultured photomixotrophically at <TEX>$80{\mu}mol\;photons\;m^{-2}s^{-1}$</TEX>, by two sides. Cells are grown in TAP medium as aerobic stage for culture growth; in logarithmic phase cells are transferred from aerobic to an anaerobic and sulfur deprived TAP- S medium and 12 mg/L initial chlorophyll content for <TEX>$H_2$</TEX> production which is monitored by the water columns and later detected by Gas Chromatography. Total produced hydrogen was <TEX>$82{\pm}10$</TEX>, <TEX>$180{\pm}20$</TEX>, <TEX>$196{\pm}20$</TEX>, <TEX>$290{\pm}30mL$</TEX> for CC124, D240, D239-40, D240-41, respectively. <TEX>$H_2$</TEX> production rates for mutant strains was <TEX>$1.3{\pm}0.5mL/L.h$</TEX> meanwhile CC124 showed 2-3 fold lower rate as <TEX>$0.57{\pm}0.2mL/L.h$</TEX>. Hydrogen production period was <TEX>$5{\pm}2days$</TEX> for CC124 and mutants showed a longer production time for <TEX>$9{\pm}2days$</TEX>. It is seen from the results that <TEX>$H_2$</TEX> productions for mutant strains have a significant effect in terms of productivity, yield and production time.