Development Of Microbial Cell Factories Research Articles

Machine Learning-Guided Optimization of p-Coumaric Acid Production in Yeast.

Industrial biotechnology uses Design-Build-Test-Learn (DBTL) cycles to accelerate the development of microbial cell factories, required for the transition to a biobased economy. To use them effectively, appropriate connections between the phases of the cycle are crucial. Using p-coumaric acid (pCA) production in Saccharomyces cerevisiae as a case study, we propose the use of one-pot library generation, random screening, targeted sequencing, and machine learning (ML) as links during DBTL cycles. We showed that the robustness and flexibility of the ML models strongly enable pathway optimization and propose feature importance and Shapley additive explanation values as a guide to expand the design space of original libraries. This approach allowed a 68% increased production of pCA within two DBTL cycles, leading to a 0.52 g/L titer and a 0.03 g/g yield on glucose.

Improving Microbial Cell Factory Performance by Engineering SAM Availability.

Methylated natural products are widely spread in nature. S-Adenosyl-l-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

The HpSGNi system: A compact approach for genetic suppression without sequence limitation in Escherichia coli

Targeted gene regulation is indispensable for exploring gene functions in microbes and the development of microbial cell factories. While most loci can be regulated by CRISPRi, it cannot be used for targets lacking protospacer adjacent motifs (PAM) or protospacer flanking sequences (PFS). Here, we characterized a genetic suppression approach named the hpDNA-assisted structure-guided nuclease mediating interference system (HpSGNi). It was composed of a flap endonuclease 1 (FEN1) and mis-hairpin DNA probes (mis-hpDNA) to suppress the expression of target genes simply and efficiently in microbe without sequence restrictions. By inhibiting the initiation and elongation of the transcription, HpSGNi successfully silenced the transcription of exogenous fluorescent protein genes, ampicillin resistance gene and endogenous folP/sulA genes in Escherichia coli BL21(DE3) and K-12 MG1655. Meanwhile, aiming at optimizing the mis-hpDNA, we displayed the characteristics by detecting the tolerance to the single base mismatch and length of the guide sequence. This DNA-guided recognition platform provides a simple approach for selectively inhibiting gene expression.

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system

BackgroundAromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes.ResultsTo facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L.ConclusionsWe developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli.

Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid-liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2'-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux.

Yeasts Have Evolved Divergent Enzyme Strategies To Deconstruct and Metabolize Xylan.

Together with bacteria and filamentous fungi, yeasts actively take part in the global carbon cycle. Over 100 yeast species have been shown to grow on the major plant polysaccharide xylan, which requires an arsenal of carbohydrate active enzymes. However, which enzymatic strategies yeasts use to deconstruct xylan and what specific biological roles they play in its conversion remain unclear. In fact, genome analyses reveal that many xylan-metabolizing yeasts lack expected xylanolytic enzymes. Guided by bioinformatics, we have here selected three xylan-metabolizing ascomycetous yeasts for in-depth characterization of growth behavior and xylanolytic enzymes. The savanna soil yeast Blastobotrys mokoenaii displays superior growth on xylan thanks to an efficient secreted glycoside hydrolase family 11 (GH11) xylanase; solving its crystal structure revealed a high similarity to xylanases from filamentous fungi. The termite gut-associated Scheffersomyces lignosus, in contrast grows more slowly, and its xylanase activity was found to be mainly cell surface-associated. The wood-isolated Wickerhamomyces canadensis, surprisingly, could not utilize xylan as the sole carbon source without the addition of xylooligosaccharides or exogenous xylanases or even co-culturing with B. mokoenaii, suggesting that W. canadensis relies on initial xylan hydrolysis by neighboring cells. Furthermore, our characterization of a novel W. canadensis GH5 subfamily 49 (GH5_49) xylanase represents the first demonstrated activity in this subfamily. Our collective results provide new information on the variable xylanolytic systems evolved by yeasts and their potential roles in natural carbohydrate conversion. IMPORTANCE Microbes that take part in the degradation of the polysaccharide xylan, the major hemicellulose component in plant biomass, are equipped with specialized enzyme machineries to hydrolyze the polymer into monosaccharides for further metabolism. However, despite being found in virtually every habitat, little is known of how yeasts break down and metabolize xylan and what biological role they may play in its turnover in nature. Here, we have explored the enzymatic xylan deconstruction strategies of three underexplored yeasts from diverse environments, Blastobotrys mokoenaii from soil, Scheffersomyces lignosus from insect guts, and Wickerhamomyces canadensis from trees, and we show that each species has a distinct behavior regarding xylan conversion. These findings may be of high relevance for future design and development of microbial cell factories and biorefineries utilizing renewable plant biomass.

Metabolic engineering of Escherichia coli for biological production of 1, 3-Butanediol

The production of 1,3-butanediol (1,3-BDO) from glucose was investigated using Escherichia coli as the host organism. A pathway was engineered by overexpressing genes phaA (acetyl-CoA acetyltransferase), phaB (acetoacetyl-CoA reductase), bld (CoA-acylating aldehyde dehydrogenase), and yqhD (alcohol dehydrogenase). The expression levels of these genes were optimized to improve 1,3-BDO production and pathways that compete with 1,3-BDO synthesis were disrupted. Culture conditions were also optimized, including the C: N ratio, aeration, induction time, temperature, and supplementation of amino acids, resulting in a strain that could produce 1,3-BDO at 257 mM in 36 h, with a yield of 0.51 mol/mol in a fed-batch bioreactor experiment. To the best of our knowledge, this is the highest titer of 1,3-BDO production ever reported using biological methods, and our findings provide a promising strategy for the development of microbial cell factories for the sustainable synthesis of other acetyl-CoA-derived chemicals.

How do engineered Yarrowia lipolytica strains secrete free fatty acids: hints from comparative transcriptomics.

Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.

Dual-plasmid interactions stimulate the accumulation of valencene in Saccharomyces cerevisiae

Plasmids are one of the most commonly used basic tools in the construction of microbial cell factories, the use of which individually or in pairs play an important role in the expression of exogenous gene modules. However, little attention has been paid to the interactions of plasmid-plasmid and plasmid-host in the widespread use of the double plasmid system. In this study, we demonstrated that dual-plasmid interactions facilitated to cell growth and product accumulation in Saccharomyces cerevisiae. The strain containing both the expression plasmid pEV (a plasmid carrying the gene encoding valencene synthase) and the assistant plasmid pI (an empty plasmid expressing no extra gene) showed a significant improvement in relative growth rate, biomass and valencene production compared to the strain containing only the pEV plasmid. The transcriptional level analysis revealed an up-regulated expression of specific gene on the expression plasmid pEV stimulated by the assistant plasmid pI in the dual-plasmid interactions. Further investigations demonstrated the essential roles of the promoters of the expression plasmid pEV and the CEN/ARS element of the assistant plasmid pI in the dual-plasmid interactions. Combined with the results of predicted nucleosome occupancy, a response model of interaction based on the key T(n)C and CEN/ARS element was established. Moreover, the transformation order of the two plasmids significantly affected the response effect, implying the dominance of plasmid pI in the dual-plasmid interactions. Our finding first demonstrated that dual plasmids regulate the gene expression through spatial interactions at DNA sequences level, which provides a new perspective for the development of microbial cell factories in future.

Exploring the potential of novel R. kratochvilovae red yeasts towards the sustainable synthesis of natural carotenoids

The development of microbial cell factories is nowadays a main route for the production of safe and environmentally friendly bioactive compounds. Rhodosporidium kratochvilovae yeasts constitute an unexplored source for the production of natural carotenoids, thus further insights on their metabolism and fermentative behavior would contribute towards the sustainable synthesis of bioactive compounds. The aim of this study was to investigate the ability of two R. kratochvilovae isolates (FMCC Y-42 and Y-43) to produce carotenoids under different experimental conditions. Cell growth, carotenoid synthesis and lipid accumulation were evaluated using synthetic media containing either glucose or mixtures of glucose:galactose (1:1), under different carbon-to-nitrogen (C/N) ratios. Carotenogenesis was highly affected by C/N ratio in both strains, while a competitive trend with lipid accumulation was also observed. Yeast strain Y-42 reached the highest concentration of total carotenoids (2.59 mg/L) when grown on glucose-based medium at a C/N ratio of 80 after 39 h of fermentation. Likewise, the strain Y-43 produced approximately 2 mg/L of total carotenoids from both glucose (C/N of 80) and its mixture with galactose (C/N 80 and 120), with similar biomass concentration (9.3, 10.3 and 10.9 g/L, respectively). HPLC-DAD analysis of carotenoid extracts indicated that the main carotenoid was β-carotene (60–75%) followed by γ-carotene (9–18%), torulene (6–13%) and torularhodin (5–17%), regardless the fermentation conditions or the strain-employed. This is the first study to reveal γ-carotene synthesis by R. kratochvilovae strains. The obtained results provided new knowledge on the production of natural carotenoids by novel red yeast strains and could pave the way towards their application in sustainable functional food products and nutraceuticals.

Biosynthesis of mushroom-derived type II ganoderic acids by engineered yeast

Type II ganoderic acids (GAs) produced by the traditional medicinal mushroom Ganoderma are a group of triterpenoids with superior biological activities. However, challenges in the genetic manipulation of the native producer, low level of accumulation in the farmed mushroom, the vulnerabilities of the farming-based supply chain, and the elusive biosynthetic pathway have hindered the efficient production of type II GAs. Here, we assemble the genome of type II GAs accumulating G. lucidum accession, screen cytochrome P450 enzymes (CYPs) identified from G. lucidum in baker’s yeast, identify key missing CYPs involved in type II GAs biosynthesis, and investigate the catalytic reaction sequence of a promiscuous CYP. Then, we engineer baker’s yeast for bioproduciton of GA-Y (3) and GA-Jb (4) and achieve their production at higher level than those from the farmed mushroom. Our findings facilitate the further deconvolution of the complex GA biosynthetic network and the development of microbial cell factories for producing GAs at commercial scale.

Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6g L-1 of FOS, with a productivity of 1.53 ± 0.03g L-1h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented.

An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L−1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.

GEDpm-cg: Genome Editing Automated Design Platform for Point Mutation Construction in Corynebacterium glutamicum.

Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.

An Integrative Toolbox for Synthetic Biology in Rhodococcus.

The development of microbial cell factories requires robust synthetic biology tools to reduce design uncertainty and accelerate the design-build-test-learn process. Herein, we developed a suite of integrative genetic tools to facilitate the engineering of Rhodococcus, a genus of bacteria with considerable biocatalytic potential. We first created pRIME, a modular, copy-controlled integrative-vector, to provide a robust platform for strain engineering and characterizing genetic parts. This vector was then employed to benchmark a series of strong promoters. We found PM6 to be the strongest constitutive rhodococcal promoter, 2.5- to 3-fold stronger than the next in our study, while overall promoter activities ranged 23-fold between the weakest and strongest promoters during exponential growth. Next, we used an optimized variant of PM6 to develop hybrid-promoters and integrative vectors to allow for tetracycline-inducible gene expression in Rhodococcus. The best of the resulting hybrid-promoters maintained a maximal activity of ∼50% of PM6 and displayed an induction factor of ∼40-fold. Finally, we developed and implemented a uLoop-derived Golden Gate assembly strategy for high-throughput DNA assembly in Rhodococcus. To demonstrate the utility of our approaches, pRIME was used to engineer Rhodococcus jostii RHA1 to grow on vanillin at concentrations 10-fold higher than what the wild-type strain tolerated. Overall, this study provides a suite of tools that will accelerate the engineering of Rhodococcus for various biocatalytic applications, including the sustainable production of chemicals from lignin-derived aromatics.

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms

The heavy global dependence on petroleum-based industries has led to serious environmental problems, including climate change and global warming. As a result, there have been calls for a paradigm shift towards the use of biorefineries, which employ natural and engineered microorganisms that can utilize various carbon sources from renewable resources as host strains for the carbon-neutral production of target products. C4 alcohols are versatile chemicals that can be used directly as biofuels and bulk chemicals and in the production of value-added materials such as plastics, cosmetics, and pharmaceuticals. C4 alcohols can be effectively produced by microorganisms using DCEO biotechnology (tools to design, construct, evaluate, and optimize) and metabolic engineering strategies. In this review, we summarize the production strategies and various synthetic tools available for the production of C4 alcohols and discuss the potential development of microbial cell factories, including the optimization of fermentation processes, that offer cost competitiveness and potential industrial commercialization.

Recent Advances in Systems Metabolic Engineering Strategies for the Production of Biopolymers

Biopolymers consisting of at least one monomer, which are produced from renewable carbon sources, are being highly sought out since ubiquitous plastics are mainly produced from petrochemical processes causing severe environmental pollution. Therefore, the development of microbial cell factories, which can efficiently synthesize diverse types of monomers and polymers, is also becoming increasingly important. The applicability of traditional metabolic engineering strategies has extended with the combination of systems biology, synthetic biology, and evolutionary engineering in a systemic and versatile manner, and are collectively termed as systems metabolic engineering. Accordingly, recent advances in biotechnology have paved the way for enabling the production of an increasing number of monomers and polymers by providing several tools and strategies associated with systems metabolic engineering. In this review, we have focused on the substantial efforts made on the development of different approaches of systems metabolic engineering, particularly based on synthetic biology and evolutionary engineering, for the efficient production of monomers and polymers.

From plant biomass to biofuels and bio-based chemicals with microbial cell factories

Global energy demands and global warming represent key challenges of the future of human society. Continous renewable energy supply is key for sustainable economy development. Waste plant biomass represent abundant source of renewable energy that can be transformed to biofuels and other value-added products, which is currently limited due to the lack of cost-effective biocatalysts. The bottleneck of this process is the degradation of structural polysaccharides of plant cell walls to soluble compounds that can be fermented to solvents or transformed to biogas via methanogenesis and can be used as biofuels or chemical raw materials. In order to replace traditional physical and chemical methods of lignocellulose pretreatment with more environmentally friendly biological approaches, native microbial enzyme systems are increasingly being explored as potential biocatalysts that could be used in these processes. Microbial enzymes are useful either as catalysts in the enzymatic hydrolysis of lignocelluloses or as components incorporated in engineered microbes for consolidated bioprocessing of lignocelluloses. The unprecedented development of tools for genetic and metabolic engineering for a wide range of microorganisms enabled significant progress in the development of microbial cell factories optimized for the producton of biofuels. One of the most promising strategies aimed towards this goal, i.e. systematic design and heterologous expression of »designer cellulosomes« in industrial solventogenic strains is adressed in detail.

Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina

Pseudomonas mendocina NK-01 previously isolated by our lab is able to accumulate medium-chain-length polyhydroxyalkanoate (mcl-PHA) intracellularly and secrete alginate oligosaccharide (AO) to the extracellular milieu. The present study aimed at investigating whether improved production of mcl-PHA and AO by P. mendocina can be accomplished by genome reduction. In this study, 14 large genomic fragments accounting for 7.7% of the genome of P. mendocina NK-01 were sequentially deleted to generate a series of genome-reduced strains by an upp-based markerless knockout method. As a result, the intracellular ATP/ADP ratio of the strain NKU421 with the largest deletion improved by 11 times compared to NK-01. More importantly, the mcl-PHA and AO yields of NKU421 increased by 114.8% and 27.8%, respectively. Enhancing mcl-PHA and AO production by NKU421 may be attributed to improved transcriptional levels of PHA synthase genes and AO secretion-related genes. The present study suggests that rational reduction of bacterial genome is a feasible approach to construct an optimal chassis for enhanced production of bacterial metabolites. In the future, further reduction of the NKU421 genome can be expected to create high-performance chassis for the development of microbial cell factories.