Increased Biomass Yield of Lactococcus lactis by Reduced Overconsumption of Amino Acids and Increased Catalytic Activities of Enzymes

Kaarel Adamberg,Andrus Seiman,Raivo Vilu,Joshua L Heazlewood

doi:10.1371/journal.pone.0048223

Kaarel Adamberg, Andrus Seiman + Show 2 more

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https://doi.org/10.1371/journal.pone.0048223

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Abstract

Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of Lactococcus lactis from 0.10 to 0.12 C-mol C-mol−1 with an increase in specific growth rate by 5 times from 0.1 to 0.5 h−1. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h−1 followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h−1). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h−1). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions.

Highlights

Lactococcus lactis is widely used in food- and biotechnological processes because the American Food and Drug Administration (FDA) classified it as GRAS in the Food Additives Amendment of 1958
Based on an annotated network taken from the BioCyc database collection, only four amino acids cannot be synthesized by L. lactis IL1403, no published experimental results confirm this statement
Growth of Lactococcus lactis is only possible in the presence of an external supply of several nutrients such as sugars, amino acids, vitamins and nucleobases, whose availability determine the cell physiology and specific growth rate

Summary

Introduction

Lactococcus lactis is widely used in food- and biotechnological processes because the American Food and Drug Administration (FDA) classified it as GRAS in the Food Additives Amendment of 1958 It is used in dairy starter cultures and there have been several attempts to use lactococci as cell factories for the production of various compounds (flavors, bacteriocins, vitamins etc.) [1]. The highest growth yields and growth rates obtained in defined media containing all amino acids remain 15 to 30% lower than those obtained in complex media such as M17 [7] This can be explained by additional cofactors (vitamins) or nucleosides present in the M17 medium in addition to more efficient transport of peptides in M17 compared to amino acid transport in defined media [8]. Poolman and Konings [4] show that the uptake Km for valine is 12 mM while the Km for leucine is 6 mM despite the fact that both amino acids are transported by the same permease

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