Abstract Lactococcus lactis, a homofermentative lactic acid bacterium, is widely applied in fermentation industry. In this study, a reductive TCA pathway was constructed in L. lactis for the biosynthesis of malic acid, a C4-dicarboxylic acid platform molecule. First, genes encoding malate dehydrogenase (mdh) from Actinobacillus succinogenes and pyruvate carboxylase (pycAY715T) from L. lactis were expressed in L. lactis NZ9000 to establish the reductive TCA pathway. The gene encoding lactate dehydrogenase (ldh) was simultaneously deleted. Malic acid was accumulated in the resulting strain NZ02-1 with a titer of 1.92 g/L. Subsequently, genes including ldhB, ldhX, als, and pfl, were deleted, increasing the titer to 3.13 g/L in strain NZ03. A promoter library was developed based on the core region of the phosphofructose kinase promoter Ppfk, and a stronger, constitutive promoter was screened for the enhanced expression of pycAY715T, leading to an increased titer of 5.75 g/L in strain NZ20. Through fed-batch fermentation, the titer in strain NZ20 was boosted to 14.1 g/L with a yield of 0.19 g/g glucose. Through a dual-phase fermentation, the titer was improved to 20.52 g/L with a yield of 0.29 g/g. Our study enabled the biosynthesis of malic acid in L. lactis, expanding the application of this strain as a chassis strain in fermentation industry.

中文翻译：

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通过建立还原性 TCA 途径和启动子工程使乳酸乳球菌中苹果酸的生物合成成为可能

摘要 乳酸乳球菌是一种同型发酵乳酸菌，广泛应用于发酵工业。在这项研究中，在乳酸乳球菌中构建了一条还原性 TCA 途径，用于苹果酸（一种 C4-二羧酸平台分子）的生物合成。首先，编码来自产琥珀酸放线杆菌的苹果酸脱氢酶 (mdh) 和来自乳酸乳球菌的丙酮酸羧化酶 (pycAY715T) 的基因在乳酸乳球菌 NZ9000 中表达，以建立还原性 TCA 途径。编码乳酸脱氢酶 (ldh) 的基因同时被删除。苹果酸在所得菌株NZ02-1中累积，滴度为1.92g/L。随后，包括 ldhB、ldhX、als 和 pfl 在内的基因被删除，使 NZ03 菌株的滴度增加到 3.13 g/L。基于磷酸果糖激酶启动子 Ppfk 的核心区域开发了启动子文库，筛选出更强的组成型启动子以增强 pycAY715T 的表达，导致菌株 NZ20 中的滴度增加 5.75 g/L。通过补料分批发酵，NZ20 菌株的效价提高到 14.1 g/L，葡萄糖产量为 0.19 g/g。通过双相发酵，滴度提高到20.52 g/L，产量为0.29 g/g。我们的研究使乳酸菌中苹果酸的生物合成成为可能，扩大了该菌株在发酵工业中作为底盘菌株的应用。29 克/克。我们的研究使乳酸菌中苹果酸的生物合成成为可能，扩大了该菌株作为发酵工业中的底盘菌株的应用。29 克/克。我们的研究使乳酸菌中苹果酸的生物合成成为可能，扩大了该菌株在发酵工业中作为底盘菌株的应用。