Enhancement of Sulfur Conversion Rate in the Production of l-Cysteine by Engineered Escherichia coli

Abstract

Cysteine is a commercially important sulfur-containing amino acid widely used as a supplement in the agricultural and food industries. It is extremely desirable to achieve a high sulfur conversion rate in the fermentation-based cysteine production. Here, the metabolic engineering of Escherichia coli was performed to enhance the sulfur conversion rate in cysteine biosynthesis. Accordingly, the reduction of sulfur loss by the regulator decR and its yhaOM operons were deleted. serACB was integrated into chromosome with constitutive promoter to coordinately increase sulfur utilization. The sulfur assimilation pathways and sulfur transcriptional regulator cysB were overexpressed to regulate sulfur metabolism and enhance sulfur conversion significantly. After the process optimization in fed-batch fermentation, LH16 [SLH02 ΔyhaM Ptrc1-serACB-cysM-nrdH-(pLH03, pTrc99a-cysB)] produced 7.5 g/L of cysteine with a sulfur conversion rate of 90.11%. These results indicate that cysteine production by LH16 is a valuable process in the agricultural and food industries.

Fitness of chassis cells and metabolic pathways for L-cysteine overproduction in Escherichia coli

“…Fluorescence intensity was monitored by microplate reader (BioTek Instruments, Winooski, VT, USA). Cys was analyzed with precolumn derivatization by HPLC (LC-10AT, Shimadzu, Japan) using a WondalSil C18 column (GL Sciences Inc., Japan) as described in our previous work (Liu et al, 2018;Liu et al, 2020). The sulfur conversion rate was defined as the percentage of moles of sulfur atoms of the final amount of Cys compared with moles of total sulfur atoms in the medium.…”

“…Many microorganisms, including Escherichia coli (Liu et al, 2018;Liu et al, 2020), Corynebacterium glutamicum (Wei et al, 2019;Kondoh et al, 2019) and Pantoea ananatis (Takumi et al, 2017), have been engineered for Cys production. The highest titers of Cys ever reported in C. glutamicum and P. ananatis were about 1.0 g/L (Wei et al, 2019) and 2.2 g/L (Takumi et al, 2017), respectively.…”

“…The highest titers of Cys ever reported in C. glutamicum and P. ananatis were about 1.0 g/L (Wei et al, 2019) and 2.2 g/L (Takumi et al, 2017), respectively. However, 7.5 g/L of Cys has been achieved in E. coli (Liu et al, 2020). Over the past 20 years, four E. coli K-12 strains (BW25113, JM109, W3110, MG1655) have been applied in Cys production (Table 1).…”

“…However, these existing works focused on the modification of metabolic pathways, while ignored the fitness of chassis cells and metabolic pathways. Although LH16 achieved high titer (7.5 g/L) and sulfur conversion rate (90.1%) in fed-batch fermentation, the growth limitation of LH16 could not be well resolved (Liu et al, 2020). Simplifying the expression of metabolic pathways to balance growth and production is the key to efficient production of Cys.…”

Expanding the Structural Diversity of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols

“…In E. coli,c ysteine is biosynthesized from l-serine (Figure 3a). To increase the efficiencyo fP hC biosynthesis,w e overexpressed the endogenous E. coli native CysM under an optimized constitutive Ptrc1 promoter [27] in E. coli BL21 cells. In addition, the efficiency of ncAA biosynthesis was determined from the intracellular concentration of the O-acetyl-lserine precursor that is produced from l-serine by CysE.…”